The prevalence of obesity is higher in Western countries than in East Asian countries. It remains unknown whether microRNAs (miRNAs) are involved in the pathogenesis of the ethnic difference in obesity. The purpose of this study was to determine whether expression levels of circulating obesity-associated miRNAs are different in Europeans and Asians.The subjects were middle-aged healthy male Austrians  and Japanese . Total miRNAs in serum from each subject were analyzed using the 3D-Gene miRNA Oligo chip. miRNAs that showed significant differences between the Austrian and Japanese groups were uploaded into Ingenuity Pathway Analysis (IPA).Among 16 miRNAs that were revealed to be associated with obesity in previous studies and showed expression levels that were high enough for a reasonable comparison, serum levels of 3 miRNAs displayed significant differences between the Austrian and Japanese groups: miR-125b-1-3p was significantly lower with a fold change of −2.94 and miR-20a-5p and miR-486–5p were significantly higher with fold changes of 1.73 and 2.38, respectively, in Austrians than in Japanese. In IPA including all 392 miRNAs that showed significant differences between Austrians and Japanese, three canonical pathways including leptin signaling in obesity, adipogenesis pathway and white adipose tissue browning pathway were identified as enriched pathways.miRNAs are thought to be involved in the ethnic difference in the prevalence of obesity, which may in part be caused by different expression levels of miR-125b-1-3p, miR-20a-5p and miR-486–5p. •Ethnic difference in circulating obesity-related microRNAs (miRs) were investigated.•Levels of 2565 miRs in blood were compared between Austrians and Japanese.•miR-125b-1-3p, −20a-5p and -486–5p levels were significantly different between them.•Significant ethnic differences were found in blood levels of 392 miRs.•These miRs target the molecules comprising obesity-related three canonical pathways. The basHeritability is well recognized as an important cause of obesity. The intrapair correlation coefficients of the values for body mass index (BMI) of identical twins reared apart were reported to be 0.70 for men and 0.66 for women . The herGenetic susceptibility to obesity involves epigenetic modifications including DNA methylation and histone modification, which are influenced by age and environmental factors such as diet and physical activity . microRN2 or higher, in Western countries and East Asian countries [2, respectively. When this cutoff is used, the prevalence of obesity (BMI of 30 kg/m2 or higher) is only 2–4% in East Asian countries. From the viewpoint of prevention of type 2 diabetes and hypertension, lower (stricter) cutoffs such as 23 kg/m2 for overweight and 25 kg/m2 for obesity are recommended in East Asian countries [2 or higher) and severe obesity (BMI of 40 kg/m2 or higher) from 2017 to March 2020 were 41.9% and 9.2%, respectively [The purpose of this study was therefore to investigate the ethnic difference in obesity-related miRNAs and to explore the possibility of miRNAs as a cause of ethnic difference in the prevalence of obesity. As shown in ountries . The higountries . Accordiountries . In the ectively . In thisectively .Fig. 1Co22.1The participants in this study were healthy male Austrians (n = 20) and Japanese (n = 20). All of the Austrian participants were Caucasians, and all of the Japanese participants were originally from Japan. We tried to enroll representative healthy middle-aged Austrian and Japanese men into this study. Those receiving any medication therapy and those with histories of known inflammatory, metabolic and cardiovascular disorders or malignancy were excluded from the participants of this study. Smokers were also excluded from the participants. The Japanese participants were male healthy volunteers who were working in different districts including three cities, Nishinomiya, Sasayama or Yamagata, in Japan. The Austrian participants were recruited at the Medical University of Graz, Graz, Austria. The protocol of this study was approved by the Hyogo College of Medicine Ethics Committee (No. 3036 in 2018) and the Medical University of Graz Ethics Committee (27–166 ex 14/15). Written informed consent was provided by all of the participants. All methods were performed in accordance with the relevant guidelines and regulations.2.2Blood was collected from each participant after overnight fasting, and serum was separated. Serum samples were kept frozen at −80°C until analyses of miRNAs and measurements of leptin and adiponectin as described below.2.3Height, body weight and waist circumference were measured, and BMI and waist-to-height ratio were calculated as weight in kilograms divided by the square of height in meters and waist circumference in cm divided by height in cm, respectively. Serum leptin and adiponectin concentrations were measured by enzyme immunoassays using commercial kits, Leptin (Sandwich EIA) Human (EIA2395R) from DRG and ADIPONECTIN, ELISA, HUMAN (BVL-RD195023100-1) from BioVendor, respectively.2.4http://microrna.sanger.ac.uk/sequences/). After stringent washes, fluorescent signals were scanned with a 3D-Gene Scanner (Toray) and analyzed using 3D-Gene Extraction software (Toray). miRNA expression was normalized as follows: The raw data of each spot were substituted with a mean intensity of the background signal determined by signal intensities of all blank spots with signal intensity of the top and bottom 5%  being removed. Measurements of spots were considered to be valid when the signal intensities were greater than 2 standard deviations of the background signal intensity. The signal intensities of the valid spots were compared and the relative expression level of a given miRNA was calculated. Global normalization of the data was performed for each array, such that the median of the signal intensity was adjusted to 25. Intensity levels of 10 or higher were regarded to be high enough for analysis of comparison between the two country groups. Each miRNA level was compared after log2 transformation between the groups. Fold change in the mean value of each miRNA intensity of Austrian versus Japanese subjects was calculated as the ratio of anti-log2 values of each mean of log2-transformed data.RNA was extracted from a serum sample (300 μl) using 3D-Gene RNA extraction reagent from a liquid sample kit  according to the manufacturer's instructions as described previously . miRNA e2.5A total of 2565 miRNAs in serum were measured by using the miRNA Oligo chip. The intensities of 1744 miRNA were too low for reasonable quantitative comparison. Consequently, 821 miRNAs were available for comparison between the Austrian and Japanese groups. Among these 821 miRNAs, 392 miRNAs showed significant differences in the Austrian and Japanese groups.From previous reports , we furt2.6https://www.qiagenbioinformatics.com/products/ingenuitypathway-analysis) [The resultant miRNAs expressed in serum that showed significant differences in the Austrian and Japanese groups were analyzed using Ingenuity Pathway Analysis (IPA)  . Putativnalysis) . After lnalysis) .2.7t-test. For each of the tested miRNAs, on the basis of the observed distribution of p values, we estimated the positive false discovery rate  according to the method of Storey et al. [q values were explored with the use of Pearson's correlation coefficient. The intensity of each miRNA was transformed with the base-2 logarithm, that is, binary logarithm. We performed univariable and multivariable linear regression analyses in order to know the relationships among the three miRNAs  that were previously reported to be associated with obesity and were significantly different in Austrian and Japanese men in the present study. The intensity of each miRNA was transformed with the base-2 logarithm for normal distribution. Pearson's correlation coefficient and standardized partial regression coefficient (β) were calculated in the univariable and multivariable analyses, respectively. In multivariable linear regression analysis for the relationship between two miRNAs out of miR-125b-1-3p, -20a-5p and -486–5p, adjustment was performed by using levels of the miRNA (as the other explanatory variable for adjustment) other than the above two paired miRNAs (an explanatory variable and an independent variable). Thus, only two variables of miRNA were used as the explanatory variables, and no other variables were used for adjustment in multivariable linear regression analysis. All p values were two-sided, and p values less than a significance level of 0.05 were considered statistically significant. All q values were two-sided, with statistical significance determined by a false discovery rate of less than 0.05. Data were analyzed with the use of SPSS version 25.0 Armonk, NY, USA.Continuous variables are summarized as means with standard deviations and were compared with the use of Welch's corrected y et al. . Associa33.13.23.3p = 0.19); between miR-125-1-3p and miR-486–5p levels, −0.669 (p < 0.01); between miR-20a-5p and miR-486–5p levels, 0.761 (p < 0.01).3.4p-value filter for pathway significance of p < 0.05, 366 canonical pathways were identified as enriched pathways in the dataset of the miRNAs showing significant differences in the Austrian and Japanese groups. These canonical pathways include leptin signal in obesity  that showed significant differences in microarray expression analysis. Using a 1.79E-05 ), adipog2.52E-07 ), and wh1.73E-04 ).Fig. 3O4The expression levels of 16 circulating miRNAs, which were reported as increased or decreased in obese individuals, were compared in the Austrian and Japanese subjects: miR-125b-1-3p was lower and miR-20a-5p and miR-486–5p were higher in the Austrian group than in the Japanese group and were thus suggested to be associated with the ethnic difference in the prevalence of obesity. This study is, to the best of our knowledge, the first study showing an ethnic difference in obesity-related miRNAs. On the other hand, among the 16 miRNAs tested, miR-221–3p, -223–3p and -486–3p levels were not different in the two groups and miR-103a-3p and -423–5p levels were significantly higher in the Austrian group than in the Japanese group, and this direction of changes in miRNAs was opposite to that shown in previous studies on obesity-associated miRNAs ,20,22,27By using IPA, we further explored the canonical pathways that were targeted by all 392 miRNAs displaying significant differences in the Austrian and Japanese subjects. As a result, three obesity-related pathways, including leptin signaling in obesity, adipogenesis pathway, and white adipose tissue browning pathway, were found associated with the above 392 miRNAs. These miRNAs were suspected to target the molecules including STAT3, PI3K, AKT and FOXO1 in leptin signal in obesity, PPARγ and FOXO1 in adipogenesis pathway, and p38MAPK, PPARγ and PPARα in white adipose tissue browning pathway , Fig. 5.In this study, three circulating miRNAs, miR-125b-1-3p, -20a-5p and -486–5p, which are included in the reported biomarkers for obesity, were associated with the ethnic difference in obesity of Austrian and Japanese men. miR-125b has been shown to target the 3′-UTR of the mRNA of phosphoinositide 3-kinase catalytic subunit δ (PI3KCD), the expression level of which was reported to be decreased in hepatocytes in obese mice . Therefoβ = 0.761). This finding appears plausible since both miR-20a-5p and miR-486–5p are involved in adipogenesis as mentioned above [The results of linear regression analysis suggest that miR-486–5p levels were associated with miR-125b-1-3p and miR-20a-5p levels, while there was no association between miR-125b-1-3p and miR-20a-5p levels. miR-20a-5p levels showed a strong correlation with miR-486–5p levels, which was independent of miR-125b-1-3p levels in multivariable analysis  in either the Austrian group or Japanese group. In addition, the percentages of subjects with an upper category of overweight (BMI of 27.5 kg/m2 or higher) were 15% in the Austrian group and 10% in the Japanese group. Thus, one possible reason for the above discrepancy is that most of the Austrian and Japanese subjects in this study did not have high BMI. Interestingly, Kuo and Halpern reported that there was no association between BMI and blood adiponectin levels in healthy adults. They speculated that obesity-related changes in adiponectin levels in previous studies were a consequence of obesity-related metabolic disorders [Although weight and BMI were larger and higher, respectively, in the Austrian group than in the Japanese group, serum leptin and adiponectin levels were comparable in the two groups. In this study, there were no subjects with obesity . Therefore, future studies using data from obese Austrian and Japanese participants would be interesting to know ethnic differences in miRNA expression in obese individuals.There are limitations of this study. In this study, serum levels of 2565 miRNAs were measured by microarray analysis without amplification; however, only about one third of the total miRNAs showed intensity levels that were high enough for comparison and thus could be analyzed for comparison. Therefore, further studies using measurements with PCR amplification are needed to compare other approximately two thirds of miRNAs in different ethnicities. The subjects of this study were all men, and it is thus necessary to test the ethnic difference in circulating miRNAs in women. Only miRNA expression was evaluated in this study, and simultaneous evaluation of mRNA expression is needed in future studies to clarify molecules that are targeted by miRNAs showing ethnic difference. As candidates of miRNAs explaining the ethnic difference in obesity, we included miRNAs that were shown to differ in obese and non-obese subjects in children as well as adults . HoweverIn summary, differences in miRNA expression levels in blood were totally analyzed and compared in Austrian and Japanese men. Circulating miR-125b-1-3p, miR-20a-5p and miR-486–5p levels were significantly different between the Austrian and Japanese subjects. By IPA, we provide evidence for an impact of the ethnic differences in the expression of 392 miRNAs on three obesity-related canonical pathways, including leptin signaling in obesity, adipogenesis pathway, and white adipose tissue browning pathway. Thus, miRNAs are thought to partly explain the difference in obesity prevalence in East Asian and Western countries. Future studies are needed to determine the molecules that are regulated by the miRNAs causing the ethnic difference in obesity and the significance of their levels in blood in relation to the pathogenesis of obesity.Grant-in-Aid for Scientific Research (No. 17H02184) from the 10.13039/501100001691Japan Society for the Promotion of Science (to IW).This study was supported by a The authors declare no competing interests.Ichiro Wakabayashi: Conceptualization, Data curation, Methodology, Investigation, Visualization, Writing – original draft, Funding acquisition. Yoko Sotoda: Data curation, Investigation. Klaus Groschner: Investigation, Visualization, Writing – review & editing. Peter P. Rainer: Data curation, Investigation. Harald Sourij: Conceptualization, Data curation, Investigation, Writing – review & editing.


We described the relationship between extreme weather-related events and death, globally, from 1999 through 2018. We used data from the emergency events database of the Université Catholique de Louvain. We also categorized the countries’ income according to the World Bank GDP and we used the CO2 emission levels data from the Carbon Dioxide Information Analysis Center to link the GDP and CO2 emissions to years of extreme weather conditions in each country. We conducted descriptive and Poisson Regression analysis to analyze the data. A total of 77 countries reported 425 extreme weather-related events from1999 through 2018. Mortality related events were highest in middle-income countries due to severe winter conditions  and cold-waves . The total number of recorded deaths due to heat waves was highest in high-income countries . Furthermore, the number of deaths in high-income countries, compared to low-income countries, was five-fold higher . The mortality rate in heat season was almost seven-fold higher than that in cold/severe winter . The number of deaths increased significantly with the repetition of extreme events . We found the number of deaths increased in high-income countries, and this was associated with an increase in the number of times extreme events occurred per year and with heat wave.Due to rising temperatures and CO A studyIncreasing greenhouse gases affects temperature, which can contribute to extreme weather-related events . Most st22.12.1.1Temperature data and number of deaths for all international events of heat wave, cold wave, and severe winter condition from 1999 to 2018 were obtained from the Emergency Events Database (EM-DAT) of the Université Catholique de Louvain, located in Brussels, Belgium .The EM-DAT was launched in 1988 as a humanitarian database, and it has collected data on > 21,000 disasters that have occurred globally since 1900. The classification of disasters, in EM-DAT, is based on the Integrated Research on Disaster Risk Peril Classification and Hazard Glossary .The EM-DAT data sources include United Nations agencies, governments, the International Federation of Red Cross and Red Crescent Societies, other non-governmental organizations, insurance companies, research institutions, and press agencies .Inclusion criteria: One or more of the following criteria must be fulfilled for a weather event to be considered a disaster event in the EM-DAT: (1) ≥ 10 people died, (2) ≥ 100 people were affected, (3) the declaration of a state of emergency or (4) a call for international assistance. Each of the severe weather events reported in this study was qualified as a disaster event. A detailed description of the EM-DAT structure, classification, variable descriptions, and definitions is available under EM-DAT: https://www.emdatbe/guidelines.2.1.22 emission levels data, which was measured as CO2 emissions (Kt) from the burning of fossil fuels, was downloaded from the Carbon Dioxide Information Analysis Center .The countries’ income category was determined based on the World Bank Gross Domestic Product (GDP)  informat2.1.3The definitions used by EM-DAT were as follow:Extreme temperature: a general term for temperature variations above (extreme heat) or below (extreme cold) normal conditions.Heat wave: a period of abnormally hot and/or unusually humid weather.Cold wave: a period of abnormally cold weather. Both weather extremes typically last two or more days. Severe winter conditions include snow, ice, frost, and freeze  for the events of deaths due to extreme weather conditions .First, we conducted descriptive analysis—which included the mean, median, minimum, maximum, and inter quartile range—to determine the number of deaths from each weather-related event, as well as temperature and COEthical approval was not required, as this study did not include human subjects.3Our descriptive analysis showed that the total number of extreme weather-related events from 1999 through 2018 was 422 in 77 countries. The distribution of these events was 15 (4%), 227 (53%), and 183 (43%) for low-, middle-, and high-income countries, respectively .Table 1TThe total number of recorded deaths in extreme weather-related events was 172,039. The number of deaths that occurred were 2,201 (1%), 82,334 (48%) and 87,504 (51%) in the respective order from low to high-income countries level. The number of deaths due to severe winter conditions was highest in middle-income countries  followed by high-income  and low-income countries  respectively. The total number of recorded deaths due to cold waves was 70,972 (95%) in middle-income countries, 2,748 (4%) in high-income countries, and 839 (1%) in low-income countries. The number of deaths due to heat waves was only reported in high-income  and middle-income countries ) .2 emission was 0.2Kt, 3.9Kt and 8.7Kt in low-, middle- and high-income countries, respectively (The average temperature due to severe winter conditions was lowest in high-income countries (−27.0 °C), followed by middle-income (−24.5 °C) and low-income (−18.0 °C) countries. The average temperature due to cold waves was lowest in high-income countries (–23.2 °C) . The aveectively . The numectively . The numectively .Table 2M4In this study, we reported the number of deaths due to three types of extreme weather-related events  within low-, medium-, and high-income countries. We found that the number of deaths due to heat waves was the highest in high-income countries , followed by middle-income , whereas low-income countries had no reported deaths. It seems there are risk factors in high income countries compared to LMIC that increase the risk of deaths from heat waves. The age of the population , lack ofShort-term heat acclimatization usually takes 3–12 days, but complete (long-term) acclimatization to an unfamiliar thermal environment may take several years . This meThe lack of reported deaths from heat waves in low-income countries may be due to lack of an efficient system for recording mortality information. Other studies have emphasized the importance of consistent reporting measures, especially in LMIC, especially since the mortality risk due to climate change disproportionately falls on low-income countries . Heat caThere is a growing link between climate change and non-communicable diseases (NCD). For example, extreme weather conditions due to climate change can be a growing threat for cancer patients to access their treatment facilities . Interna2 emissions due to heat-wave, cold-wave, and severe winter conditions was higher in high-income countries. Also, we found a significant correlation between the total number of deaths and CO2 emissions in cold wave events among middle-income countries. One factor which can cause more CO2 emission is urbanization.We also found that the average CO2 emissions investigated how four different aspects of urbanization affect CO2 emissions. The results showed that economic urbanization and land urbanization each have a positive impact on CO2 emissions. This impact is due to the changes in the non-built to built area and accumulation of wealth, while population and social urbanization have a negative impact on CO2 emissions , elongation of the heat wave may play a greater role in human death compared to other factors like temperature.62 emissions can result in an increase in the number of deaths during severe weather events.The results of this study show that the majority of cold wave related deaths occurred in middle-income countries followed by high-income countries. We also found that more deaths were likely to occur during heat waves than cold waves or severe winter weather, in particularly in high-income countries. Finally, increased CONo funding was received for this study.Maral Amirkhani: Conceptualization, Writing – original draft, Data curation, Writing – review & editing. Shidrokh Ghaemimood: Conceptualization, Supervision, Writing – review & editing. Johan von Schreeb: Writing – review & editing. Ziad El-Khatib: Conceptualization, Supervision, Writing – review & editing. Sanni Yaya: Conceptualization, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Quickly developing precision medicine and patient-oriented treatment strategies urgently require novel technological solutions. The randomly cell-populated scaffolds usually used for tissue engineering often fail to mimic the highly anisotropic characteristics of native tissue. In this work, an ultrasound standing-wave-based tissue engineering acoustophoretic (TEA) set-up was developed to organize murine mesenchymal stromal cells (mMSCs) in an in situ polymerizing 3-D fibrin hydrogel. The resultant constructs, consisting of 17 cell layers spaced at 300 µm, were obtained by continuous wave ultrasound applied at a 2.5 MHz frequency. The patterned mMSCs preserved the structured behavior within 10 days of culturing in osteogenic conditions. Cell viability was moderately increased 1 day after the patterning; it subdued and evened out, with the cells randomly encapsulated in hydrogels, within 21 days of culturing. Cells in the structured hydrogels exhibited enhanced expression of certain osteogenic markers, i.e., Runt-related transcription factor 2 (RUNX2), osterix (Osx) transcription factor, collagen-1 alpha1 (COL1A1), osteopontin (OPN), osteocalcin (OCN), and osteonectin (ON), as well as of certain cell-cycle-progression-associated genes, i.e., Cyclin D1, cysteine-rich angiogenic inducer 61 (CYR61), and anillin (ANLN), when cultured with osteogenic supplements and, for ANLN, also in the expansion media. Additionally, OPN expression was also augmented on day 5 in the patterned gels cultured without the osteoinductive media, suggesting the pro-osteogenic influence of the patterned cell organization. The TEA set-up proposes a novel method for non-invasively organizing cells in a 3-D environment, potentially enhancing the regenerative properties of the designed anisotropic constructs for bone healing. The approaches traditionally used in clinical top-down tissue engineering (TE), which employ three crucial components, i.e., scaffolds, cells, and biological factors , have acThere are a number of methods that attempt to reconstruct organ structure in a bottom-up manner, with one of them being cell sheet technology (CST). CST allows recreating structures of multiple cell types, albeit with limited complexity and at the cost of intensive labor, with the requirement for direct manipulation of the cell-containing material ,13. ThreThe application of external physical fields, i.e., magnetic ,17, elecIn this work, we describe a tissue engineering acoustophoretic (TEA) set-up for the organization of murine mesenchymal stromal cells (MSCs) in an in situ polymerizing fibrin hydrogel. MSCs are multipotent cells that, due to their potential for broad differentiation into chondrogenic, adipogenic, and osteogenic lineages, as well as their immunomodulatory properties, show great promise in regenerative therapies for both autologous and allogeneic transplantation . In thisIn order to design the acoustophoretic chamber with the optimal height for the constructs, the SOS in non-polymerized fibrinogen was measured, and the impact of particles, i.e., red fluorescent beads (d = 12 µm) and cells suspended in fibrinogen at a concentration of 5 mg/mL, was analyzed . The estFor the TEA experimental work, an excitation frequency of 2.5 MHz, which is equal to the resonance frequency of the piezo-crystal and enables the highest ultrasound transmission into the acoustophoretic chamber, was chosen. Given the chosen frequency and using Equation (1), we expected to receive 17 layers spaced at 300 µm in the acoustic chamber, with a height of 5.1 mm.Red fluorescent beads were patterned at the frequency of 2.5 MHz and with transmission peak-to-peak voltage (Vpp) values of 7.7, 10.6, 12.6, and 14.5 V in fibrin gels. With increasing transmission voltage, the agglomeration of beads in the constructs was observed a, and thThe established acoustic parameters were used to pattern mMSCs in fibrin gels and, as was expected, the distances corresponded to approximately 300 µm, and bands were clearly formed a. Confocal imaging of cell organization in fibrin hydrogels revealed that cells preserved the layered structure within 10 days of culturing a. FilameCellular viability was evaluated via Presto Blue assay, and a subtle but statistically significant increase in mMSC activity was observed the next day after the cell organization in hydrogels cultured in expansion degradation-free media with aprotinin (EMA) a. This eThe mMSCs were cultured either in expansion degradation-free media with aprotinin (EMA) or in osteogenic degradation-free media with aprotinin (OMA) for 5 days and compared. These two types of media were chosen to investigate the outcomes of cell patterning itself on gene expression in an osteogenic-factor-independent manner. During the differentiation of MSCs into osteoblasts, osteogenic genes experience time-dependent expression ,32. Runtp = 0.0460). However, the pairwise comparison did not confirm any statistically significant changes. The enhanced expression of osteogenic markers, i.e., ON, OPN, COL1A1, and RUNX2 , in the p = 0.0408 and p = 0.0494, respectively. However, the pairwise comparison did not result in statistical significance. The expression of early-response transcription factor JUN (c-JUN) and baculoviral IAP repeat containing 5 (Birc5) survival genes was not influenced by the cell organization. Genes influencing cell cycle progression, i.e., Cyclin D1, anillin (ANLN), and cysteine-rich angiogenic inducer 61 (CYR61), were also affected by patterning  in the op = 0.0452) of patterning on the gene expression of ALP; however, this was not observed for pairwise comparison. The degradation-free osteogenic media (OMA) was then selected for long-term culturing of the patterned constructs at additional time points. Days 7 and 10 were chosen to monitor the progression. The most pronounced effects for all genes showing elevated expression in patterned hydrogels were observed on day 5 . Osx, RUSorting cells in acoustophoretic fields is a promising method to rapidly and non-invasively organize cells at the required positions. The application of a tissue engineering acoustophoretic (TEA) set-up for the patterning of murine mesenchymal stromal cells (mMSCs) was developed in the current study to enhance the osteogenic properties of 3-D fibrin constructs for bone regeneration.The ultrasound-based TEA set-up organized mMSCs within seconds in parallel bands in an in situ polymerizing fibrin gel. The optimized acoustic parameters for the set-up were a frequency of 2.5 MHz in continuous wave mode, with a 7.7 V peak-to-peak transmission voltage. As a result of the measured SOS in fibrinogen and the selected acoustic parameters, 17 cell layers spaced at approximately 300 µm were formed. The acoustic imaging of the 3-D constructs showed a sufficiently homogenous distribution of the fluorescent beads throughout the entire scaffold c. The 10The application of ultrasound at low intensities has been shown to have a pro-regenerative influence on some soft tissues  and boneGene expression analysis revealed that certain osteogenic markers, i.e., RUNX2, OCN, OPN, Osx, ON, and COL1A1, were enhanced on at least one of the selected days . The effInterestingly, mMSCs cultured in normal expansion media without the addition of pro-osteogenic supplements also experienced a higher expression of the OPN gene on day 5 , in compDue to poor long-term cell survival in a 3-D environment, this study was limited to a 10-day culturing period. On day 15, a very low viable cell number was observed in the middle of the construct. As mentioned above, this could be overcome via the use of additional stimulatory techniques, such as ultrasound, or by culturing the constructs in perfused bioreactors. Additionally, the osteogenic differentiation was only evaluated via the gene expression of osteogenic markers, without an assessment of the properties of the formed ECM. This is planned in future experimental work, where the effect of patterning on the deposition of ECM proteins and matrix mineralization, as well as gel anisotropy, will be evaluated. The changes in the mechanical properties of the designed constructs are also planned to be tested. In this work, only relative changes in gene expression were quantified, where each data duplicate was normalized to the mean value of the non-patterned control. Since the time points were not chosen with large enough intervals, no drastic changes were observed in gene expression between the selected days. This hindered the use of absolute values for the data analysis to demonstrate the effect of cell organization and could be another limitation of this study. Most certainly, the TEA set-up described here cannot be directly used for the creation of 3-D bone regenerative constructs, due to the complex hierarchy of bone structure. However, this set-up can be combined in the future with other high-precision techniques, e.g., 3-D bioprinting ,70,71, tThe TEA set-up a consistFibrin hydrogel components and beads/cells were added into the acoustophoretic chamber b, givingAs a result of acoustophoresis and a reflector placed in the path of the propagating longitudinal ultrasound wave, a standing wave pattern was formed c, which 2  and stored on ice. Both gel components were equilibrated to room temperature right before adding them to the TEA set-up. The PBS used for the preparation of fibrin hydrogels did not contain antibiotics. Hydrogels were mixed in the acoustophoretic chamber: a total volume of 600 µL was received via the addition of 150 µL of thrombin to 450 µL of fibrinogen, with final concentrations of components in the gel of 1 U/mL and 5 mg/mL, respectively. To test the efficiency of acoustophoresis, red fluorescent polystyrene beads  with a diameter of 12 µm were used. For regenerative constructs, the cells were passaged as described above, centrifuged at 500 rpm for 5 min, and resuspended in fibrinogen. To ensure full polymerization, the constructs were left for an additional 50 min in the safety hood.Human fibrinogen  was dissolved in phosphate-buffered saline (PBS)  without the addition of antibiotics, then warmed up for 3 min in a water-bath at 37 °C and filtered through a 0.2 µm PVDF filter . The fibrinogen solution was stored in the incubator at 37 °C before resuspending cells or beads in it. Thrombin from bovine plasma  was dissolved in PBS containing 5 mM CaClThe acoustic pressure required for the organization of the cells was controlled via the transmission voltage at the ultrasonic transducer. For this purpose, the voltage signal was recorded on the oscilloscope via an oscilloscope probe connected to the T-piece of the amplifier output . All harDetermination of the speed of sound (SOS) in fibrin hydrogels was performed in the custom-made 3-D-printed chamber. This set-up consisted of an unfocused 5 MHz transducer emitting and detecting soundwaves, which was inserted at the bottom of a Teflon chamber containing the ultrasound transmitting media, i.e., fibrinogen at a concentration of 5 mg/mL, mimicking the initial point of the acoustophoresis. The upper part of the set-up was placed directly on top of the transducer to ensure the alignment of the cylindrical compartment with the transducer, building a cavity  for the sample medium. A steel plate covered the surface of the cavity to induce multiple reflections. SOS measurements were performed at room temperature. Double distilled water was used as a control, and values were compared to SOS ≈ 1494 m/s at room temperature (24 °C) .The transducer was connected to a US-key single-channel ultrasound device  used for the transducer excitation and digitization of the received signal. Three echoes and reflections were generated, and the time difference between the second and the third one was analyzed through cepstral analysis . Finally2, 95% humidity). On the day of producing the 3-D constructs, the cells were harvested at 70–80% confluency. For this purpose, PBS containing Pen-Strep and trypsin  were used to wash and detach the cells, respectively. Before the cells were encapsulated in fibrin gels, they were passed through a 40-µm cell strainer  and then counted to ensure no cell aggregation.Murine bone-marrow-derived mesenchymal stromal cell-line D1  was cultured in expansion media composed of high-glucose Dulbecco-modified minimal essential medium (DMEM) , 10% superior fetal bovine serum (FBS) , 1% penicillin and 1% streptomycin (Pen-Strep) , 2 mM Glutamax  in standard cultivation conditions . Osteogenic degradation-free media with aprotinin (OMA) had the same content as EMA and was additionally supplemented with osteogenic factors, i.e., 100 mM L-ascorbic acid , 50 mM β-glycerophosphate , and 5 nM dexamethasone . The media were refreshed every two days. For all experiments, mMSCs were used at a concentration of 450 cells/µL.After the gels were fully polymerized, they were taken out of the acoustophoretic chamber and washed 3 times with warm PBS. The fibrin constructs were then placed in 700 µL of expansion degradation-free media with aprotinin (EMA), which consisted of low-glucose DMEM media with sodium pyruvate , 10% FBS, 2 mM Glutamax, Pen-Strep, 5 mM CaClv/v with the expansion media and added to the fibrin gels. The gels containing the reagent were incubated for 4 h at 37 °C, shaking at 150 rpm . The absorbance was measured in duplicate at 570 nm with a reference wavelength of 600 nm on Tecan .The Presto-Blue reagent  was mixed 1:10 The constructs were washed in PBS and fixed for 20 min in a 4% paraformaldehyde (PFA) solution . The gels were cut into 3 vertical slices similar in size to observe the cell layers. The cells were then permeabilized with 0.1% Triton X-100  for 10 min, followed by two rounds of washes in PBS. Fibrous actin was stained for 2 h by phalloidin-tetramethylrhodamine B isothiocyanate  prepared in PBS containing 1% bovine serum albumin  at a 1:40 ratio. The gels were then again washed 3 times in PBS. Nuclei were counterstained with Hoechst 33,542  at 1:200 in PBS with 1% BSA for 2 h. The gels were finally washed 3 times in PBS and imaged either on a Leica TCS SP5 confocal microscope  or on an inverted AxioObserver  fluorescent microscope. All of the incubation steps were performed at room temperature at 150 RPMs on the microplate shaker . Images were processed and analyzed using Image-J software version 1.53s .The custom-built SAM-200Ex set-up  was used® scalpel  and submerged into the lysis buffer; they were kept at −20 °C until frozen. Then, the gels were thawed and pipetted up and down rigorously. The isolated mRNA was transcribed to complementary DNA (cDNA) using qScriptTM cDNA SuperMix  on Mastercycler EP Gradient S . The gene expression was measured using SYBRTM Green PCR Master Mix  on LightCycler 480 II . The primers were designed using Primer3 Input software version 0.4.0  and produced by TIB Molbiol . The expression of all target genes was normalized to the expression of a housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primer sequences are summarized in The fibrin constructs were lysed on the selected day, and messenger RNA (mRNA) was isolated using the NucleoSpin RNA II Kit  with some modifications. To ensure efficient cell lysis, fibrin gels were disintegrated by a sterile type 11 Cutfixp ≤ 0.5. Statistical analysis was performed with GraphPad Prism software version 9.1.1 .All measurements of TEA set-up characteristics were conducted in triplicate. The biological read-outs were repeated at least three times, with each trial having at least two replicas. All data were first tested for normality using the Kolmogorov–Smirnov test, which was then followed by a t-test or a Mann–Whitney test for pairwise comparison. For comparison of more than 2 groups, one-way ANOVA followed by a Tukey multiple comparison test was performed. For two-variable analyses, a two-way ANOVA and Šidák’s multiple comparison test were used. The significance level was set at In this work, a tissue engineering acoustophoretic (TEA) set-up was developed and characterized to create 3-D anisotropic constructs with enhanced osteogenic properties for bone regeneration. The TEA set-up permits the production of 3-D building blocks for bone tissue engineering, with a high precision of cell positioning and reproducibility. The enhanced expression of osteogenic markers was observed in the TEA-patterned constructs cultured with osteogenic supplements. In addition, the expression of osteopontin (OPN) was elevated in the patterned gels even without the addition of the osteoinductive media. This quick and non-invasive method can be combined with other TE techniques, i.e., 3-D bio-printing, to create more sophisticated ex vivo constructs. Tissue complexity could also be achieved in the TEA set-up itself via changing: (i) the form of the construct, which is dependent on the selected shape in the Teflon chamber; (ii) the number of layers and the spacing length, which are dependent on the ultrasound frequency; (iii) the in situ polymerizing gel, which is dependent on the material properties; and (iv) the organization pattern, i.e., parallel planes, columns, and agglomerates, which is dependent on the number of transducers used. Methods permitting the long-term culturing of TEA-produced constructs, i.e., ultrasound-based stimulation and perfusion, are also in need of being further developed.


In COVID-19 patients, the platelet count correlated with the A-aO2 gradient , with its increase over the expected , with the PaO2 values , with the PCO2 values  and with the pH values . In COVID-19 negative patients, the platelet values correlated only with the A-aO2 gradient: . Patients discharged from emergency department had a mean platelet value of 234.3 ± 68.7, those hospitalized in ordinary wards had a mean value of 204.3 ± 82.5 and in patients admitted to sub-intensive/intensive care, the mean value was 201.7 ± 75.1. In COVID-19 patients, the survivors had an average platelet value at entry to the emergency department of 220.1 ± 81.4, while that of those who died was 206.4 ± 87.7. Our data confirm that SARS-CoV-2 infection may induce thrombocytopenia, and that the reduction in platelet counts could be correlated with the main blood gas parameters and with clinical outcome; as a consequence, platelet count could be an important prognostic factor to evaluate and stratify COVID-19 patients.COVID-19 patients may manifest thrombocytopenia and some of these patients succumb to infection due to coagulopathy. The aim of our study was to examine platelet count values in patients infected with SARS-CoV-2, comparing them to a control group consisting of non-COVID-19 patients. Moreover, we evaluated the correlation between the platelet value and the respiratory alteration parameters and the outcome  in COVID-19 patients. The mean platelet values (×10 Coronavirus Disease 2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), can simultaneously manifest in different organs as well as the pulmonary system ,4. The m9/L. Data collected from blood gas analysis were as follows: partial pressure of oxygen , partial pressure of carbon dioxide , PaO2/FiO2 ratio, alveolar-arterial gradient (A-aO2 gradient). Vital parameters such as respiratory rate and peripheral oxygen saturation were also recorded.A retrospective analysis was performed on patients with suspected SARS-CoV-2 infection who referred to the Emergency Department of the Sant’Orsola-Malpighi Hospital in Bologna from March 2020 to May 2020. All patients underwent a molecular nasopharyngeal swab for the diagnosis of SARS-CoV-2 infection at entrance to the Emergency Department and were subjected to arterial blood gas analysis and blood exams. The platelet reference range was 160–370 × 10The outcome of discharge from the emergency room was also analyzed, dividing patients into those who were discharged, hospitalized in ordinary wards and hospitalized in the sub-intensive/intensive care unit. Moreover, a differentiated analysis was performed on surviving patients and those who had died,30 days after emergency department admission.p value < 0.05 was considered statistically significant. All data were collected and entered into an Excel database  and statistical analyses were performed using SPSS .Continuous variables are expressed as mean ± standard deviation. The Mann–Whitney U test for independent samples was used to compare the quantitative variables between groups. Categorical variables are presented as frequencies and percentages and compared using the Chi-squared test with Yates’ correction. The correlation between variables was measured using Pearson’s coefficient. A p < 0.001)  , with the values of PaO2   and with the pH values , while it did not correlate with peripheral O2 saturation values, respiratory rate and PaO2/FiO2 ratio. Moreover, we observed that the value of platelets inversely correlated with C-reactive protein with a statistical significance   , while no correlation with the other blood gas analysis parameters was detected. Regarding the outcome of the emergency department admission of patients with COVID-19, the discharged patients (n = 128) had a mean platelet value of 234.3 ± 68.7, those hospitalized in ordinary wards (n = 329) had a mean value of 204.3 ± 82.5 and those admitted to intensive/sub-intensive care (n = 32) had an average value of 201.7 ± 75.1. In the group of COVID-19 patients, the survivors had an average platelet value at entry of 220.1 ± 81.4, while for those who died it was 206.4 ± 87.7.A total of 998 patients were examined. A COVID-19 positive swab was detected in 489 patients, while 509 patients had negative swab (see patients’ characteristics in 001) see . In COVI149) see , with it093) see , with th −0.146) . On the  = 0.00) . In COVI2, A-aO2 gradient and its increase compared to the expected for age. Therefore, there is an inverse correlation between the degree severity of respiratory compensation and the platelet value. In particular, a direct correlation is also evident between the degree of compromise of gas exchanges at the alveolar-capillary membrane level, as evidenced by the A-aO2 gradient, and the platelet values. The fact that the value of platelets correlates with the degree of respiratory impairment could explain the finding that patients discharged to home have higher platelet values than those who are hospitalized, while patients admitted to sub-intensive or intensive care units requiring ventilatory support have the lowest values. Moreover, platelet count inversely correlated with C-reactive protein which represents an important index of systemic inflammation. On the contrary, platelet count did not correlate with procalcitonin; this aspect could be related to the specific role of procalcitonin, that represents an index of bacterial sovrainfection that is present only in some patients, especially in the case of SARS-CoV-2 infection. The platelet value detected on first medical evaluation appears to be a prognostic factor of mortality; in fact, as shown, COVID-19 patients who died within 30 days displayed lower platelet values. In published literature, several studies have demonstrated that platelets play a main role in the development of respiratory distress following lung injury. Depletion of platelets induces increased alveolar bleeding, dissemination of bacteria, plasma cytokine counts and disease severity, thus suggesting a primarily protective role for platelets [In our study, we demonstrated how platelet count values differed between the two groups (COVID-19 and non-COVID-19 patients), with lower values in COVID-19 patients. The hypothetical mechanisms by which SARS-CoV-2 causes thrombocytopenia are the following: impaired haematopoiesis caused by systemic inflammation or cytokine storm, such as IL-6 which is frequently elevated in SARS-CoV-2 infection ; SARS-Colatelets . Moreovelatelets ,12. Platlatelets . Therefolatelets . The inclatelets . These flatelets . Moreovelatelets . Severallatelets .Unfortunately, in our work, it was not possible to perform an assessment of adenosine diphosphate (ADP)-, thrombin receptor activator peptide 6 (TRAP)-, and arachidonic acid (AA)-induced platelet activity through impedance aggregometry. That method would have allowed us to further define the prognostic impact of thrombocytopenia, but the method is not widely available in an emergency room setting. Heinz and colleagues demonstrated greater fibrinolysis resistance in COVID-19 patients by thromboelastometry, but they did not find a greater platelet aggregability based on impedance aggregometric tests . A subseSARS-CoV-2 infection can induce different degrees of thrombocytopenia depending on the severity of the disease and respiratory failure. The reduction in platelet counts correlates with the main blood gas parameters of respiratory failure. Moreover, platelet count values were progressively lower depending on the severity of the clinical scenario and the degree of medical assistance required. In addition, COVID-19 subjects who died, compared to the survivors, had lower platelet values at the time of entry to the emergency room. Therefore, several etiological hypotheses can be argued from our clinical and real-life findings, but further specific studies, both in vitro and in vivo, are required to confirm those data.In conclusion, a reduction in platelet counts, albeit modest in the early stages of the disease, is one of the typical findings of SARS-CoV-2 infection and correlates with the severity of respiratory failure, representing a prognostic index of increased mortality risk.


Lung adenocarcinoma is the main pathological type of lung cancer with a very low 5-year survival rate. In the present study, through analyzing the mRNA, miRNA, and DNA methylation sequencing data from TCGA, combined with the downloaded clinical data, we accidentally found that both methylation and gene expression of some genes were up-regulated or down-regulated, which is in contradiction with our general understanding. To probe the mechanism, we selected MNDA associated with lung cancer prognosis for further analysis. The results showed that the imbalance of methylase and demethylase resulted in the demethylation of MNDA. Moreover, the expression of SPI1, the main transcription factor of MNDA, was down-regulated, while the two miRNAs hsa-miR-33a-5p and hsa-miR-33b-5p, which directly targeted MNDA, were up-regulated, thus inhibiting the expression of MNDA. In conclusion, the abnormal expression of MNDA in lung cancer is the result of the combined effects of transcriptional and post-transcriptional regulation.Lung adenocarcinoma (LA) is the main pathological type of lung cancer with a very low 5-year survival rate. In the present study, after downloading the mRNA, miRNA, and DNA methylation sequencing data from TCGA, combined with the downloaded clinical data, comparative analysis, prognostic analysis, GO and KEGG analysis, GSEA analysis, methylation analysis, transcriptional regulation and post-transcriptional regulation were performed. We found that both methylation and gene expression of MNDA in LA were down-regulated, while high expression of MNDA was associated with good overall survival in LA. To probe the mechanism, further analysis showed that SPI1 was the main transcription factor of MNDA, but it was also down-regulated in LA. At the same time, the expression of eight target miRNAs of MNDA was significantly up-regulated, and the expression of hsa-miR-33a-5p and hsa-miR-33b-5p were verified to directly target MNDA. In conclusion, the abnormal expression of MNDA in LA is the result of the combined effects of transcriptional and post-transcriptional regulation. According to the latest report of the World Health Organization (WHO) and the International Agency for Cancer Research (IARC), lung cancer is the most common malignant tumor type in human beings, accounting for 11.6% of total cancer and 18.4% of total cancer deaths ,3. Due tMyeloid cell nuclear differentiation antigen (MNDA) is a member of the immune-related hematopoietic IFN-inducible nuclear protein containing a 200-amino-acid repeat (HIN-200) family, which can act as a pattern recognition receptor to recognize foreign double-stranded DNA . The N-tIn recent years, epigenetic regulation has been increasingly considered to be an important goal of oncology . Researchttp://xena.ucsc.edu/; accessed on 20 February 2019) database to obtain the sequencing data and corresponding clinical information [http://www.cbioportal.org/; accessed on 20 February 2019). We used R language and limma package to analyze the difference between gene expression, miRNA expression and DNA methylation [p < 0.05 and log2 ≥ |1|, and the differential miRNA was screened out according to the criteria of p < 0.05 and log2 ≥ |1|. The significant differentially different genes with methylated changes were screened under the conditions of p < 0.05 and | methylation change ≥ |0.2|. All of the above data were from the TCGA database, and their use and access were based on TCGA′s data access policy and publishing guidelines . Therefore, no additional ethics committee approval was required.We used the University of California Santa Cruz (UCSC) xena  and the methylation changes at its promoter position in MNDA [https://www.proteinatlas.org/; accessed on 6 May 2022) database was used to assess protein expression of MNDA by immunohistochemistry [Paired and unpaired analytical methods were used to compare MNDA expression between LA and normal lung tissue. The UALCAN database was used to analyze the expression of MNDA protein  under the condition of Gene: MNDA; Analysis Type: Lung Cancer vs. Normal Analysis; Data Type: mRNA and ORDER BY: Under-expression: Fold Change. The expression of MNDA in published research was studied by using online database Oncomine   database was used to analyze the binding of SPI1 to the MNDA promoter and visualized with the WashU Epigenome Browser online tool  [The Encode  [The predictive target miRNAs of MNDA mRNA were selected using the online database TargetScanHuman (er 2019) .2. Total RNA was extracted from cells using TRIzol reagent . Primers were synthesized by Sangon Biotech Co., Ltd. , and the MNDA primers were 5′-ACTGACATCGGAAGCAAGAGGG-3′ (forward) and 5′-TGCAGATGTGCTGGCTCCTGAG-3′ (reverse), the SPI1 primers were 5′-GACACGGATCTATACCAACGCC-3′ (forward) and 5′-CCGTGAAGTTGTTCTCGGCGAA-3′ (reverse). Each sample was reverse-transcribed into cDNA using TransScript Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR . Then, the cDNA was synthesized by reverse transcription and amplified using 2× SYBR Green qPCR Master Mix  according to the manufacturer’s instructions. qPCR was performed at 95 °C for 3 min, and 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. GAPDH and β-actin were the internal references for the target genes. The relative expression levels of mRNA were calculated using the 2−ΔΔCT method. Human normal lung epithelial BEAS-2B cells and lung cancer cell lines PC9, H1299, and A549 cells were cultured in DMEM plus 10% fetal bovine serum (FBS) at 37 °C and 5% COThe SPI1 siRNAs and the corresponding negative controls were synthesized by Ruibo Biotechnology . 293T cells were seeded in 6-well plates for 24 h before transfection. Transfection was performed using LipofectamineTM 2000  according to the manufacturer’s instructions. The siRNA and si-NC were diluted to a concentration of 100 nmol/L, and the subsequent treatment was performed at 24 h post-transfection. The transfection efficiency was detected using RT-qPCR. 5 cells were lysed with 1 mL of RIPA buffer  containing protease inhibitors, incubated on ice for 30 min, and then centrifuged at 13,000× g for 10 min. The supernatants were collected, and quantification was performed using BCA kit. After 200 ng DNA and 100 μg protein in reaction buffer  with a final volume 20 μL were incubated for 20 min at room temperature, all of the binding reactions were loaded onto 8% non-denaturing polyacrylamide gel and run at a voltage of 100 v for the desired distance. After the retarded bands were determined by ethidium bromide, the binding of SPI1 to the promoter was detected by incubation with PU.1/SPI1 rabbit primary antibody  and corresponding secondary antibody .Primers were designed to amplify the SPI1 binding site in the MNDA promoter region, and PCR products were purified by Qiagen PCR purification kit  A total of 10The 3′-UTRs of MNDA containing hsa-miR-33a-5p, hsa-miR-33b-5p, hsa-miR-744-3p and hsa-miR-183-3p binding sites were amplified by Nanjing Genscript Biological Technology Co., Ltd and cloned into the pmirGLO vector . Then, the wild type of MNDA or mutant MNDA 3′-UTRs were constructed. Then, the constructed plasmids were co-transfected with hsa-miR-33a-5p, hsa-miR-33b-5p, hsa-miR-744-3p, hsa-miR-183-3p mimics or mimic-NC into 293T cells using Lipofectamine 2000  according to the manufacturer’s instructions. After 48 h of transfection, luciferase activities were measured by the Dual-Luciferase Reporter System .https://www.R-project.org/; accessed on 20 January 2019) using packages including Ggplot2 and Ggpubr. KM plot was performed and analyzed using a log-rank test. GraphPad Prism 8 was used for the calculation of qPCR results. Student′s t tests were used for differential expression analysis. Pearson correlation coefficients were used for correlation analysis. There was a significant difference if p value < 0.05.The statistical analysis and figure construction were completed in R software version 3.6.3  and DSS (HR = 0.68 (0.47–0.97), p = 0.035), but not to PFI (HR = 0.87 (0.67–1.13), p = 0.205), which suggested that high expression of MNDA was associated with good prognosis of LA , disease-specific survival (DSS), and progression-free interval (PFI) in LA. The results showed that MNDA was related to OS (HR = 0.72 (0.54–0.96), is of LA A–C. MNDAis of LA D.p < 0.05 and log2 ≥ |1| as the screening criteria. We obtained a total of 1278 significantly different genes, of which 541 were significantly up-regulated and 737 were significantly down-regulated. After GO analysis of the significantly different genes, it was found that biological process mainly involved leukocyte proliferation, migration, adhesion and T cell activation. Molecular function mainly involved receptor ligand activity, cytokine activity, immunoglobulin binding, G protein-coupled peptide receptor activity and MHC class II receptor activity. In addition, cellular component mainly involved external side of plasma membrane, secretory granule membrane, tertiary granule membrane, and MHC protein complex   B. In addp = 3 × 10−27)   C. High elow 251) D, so we To further confirm this conclusion, we used qPCR to detect the mRNA expression of MNDA in LA cell lines H1299, PC9 and A549, as well as in normal cell line Beas-2b. The results showed that the expression levels of MNDA in H1299, PC9 and A549 were significantly lower than that in Beas-2b. Additionally, the expression trend of SPI1 was consistent with that of MNDA E. Next, MicroRNAs (miRNAs) are a class of non-coding single-stranded RNA molecules of approximately 22 nucleotides in length encoded by endogenous genes that bind to the 3′ untranslated region (3′UTR) of mRNAs, resulting in the degradation of target mRNAs and involved in the regulation of gene expression in eukaryotic cells. We speculated that microRNAs are involved in the regulation of MNDA expression in lung cancer. Through RNAseq analysis of miRNA expression in TCGA, we found that 121 miRNAs were up-regulated and 33 miRNAs were down-regulated. We then used Targetscan Human to predict that MNDA has 59 conserved miRNAs A. IntersThe luciferase assays were carried out to detect the relationship between MNDA and hsa-miR-33a-5p, hsa-miR-33b-5p, hsa-miR-744-3p, and hsa-miR-183-3p. As illustrated in Lung cancer is one of the most commonly diagnosed cancers and the leading cause of cancer-related death worldwide, with an estimated 2 million new cases and 1.76 million deaths each year. Over the past two decades, our understanding of lung cancer biology, the use of predictive biomarkers, and improvements in therapy, especially targeted therapy and immune checkpoint inhibitors, have made remarkable progress for improved outcomes . HoweverMNDA is a nuclear protein and a stress-inducible protein, and reported studies suggest that its function is mainly related to immune cells . MNDA anWe compared the mRNA expression of MNDA between LA and normal samples in TCGA using paired and unpaired methods, and found that tumor samples had significantly lower MNDA mRNA levels than normal samples, as validated by data from Oncomine. Likewise, data from the UALCAN database and Human ALTAS database confirmed that MNDA protein was also lower in tumor samples. Further analysis showed that MNDA could be used as a prognostic biomarker for OS and DSS in LA, and as an indicator to distinguish tumor samples from normal samples. These results suggest that MNDA can be used as a novel and valuable biomarker for the diagnosis and treatment of LA.To identify the function of MNDA, we divided LA into high and low expression groups and performed differential gene analysis. The results of GO and KEGG analysis of these differential genes suggested that the functions of MNDA mainly involved leukocyte proliferation and migration, T cell activation and MHC protein complex. Using the ssGESA method, we further validated that MNDA was significantly correlated with chemokines, chemokine receptors, and MHC molecules. These results suggest that MNDA may act as a factor that activates immune infiltration in LA. However, what is puzzling is that MNDA also has a strong correlation with immune checkpoints. We speculate that it may be induced by the cooperative expression pattern between them.The regulation of gene expression is complex, delicate and multi-level. Gene expression regulation is the molecular basis of cell differentiation, morphogenesis and ontogeny in organisms. DNA methylation is an important part of epigenetics and an important mechanism for the regulation of gene expression. DNA methylation regulates gene expression by recruiting proteins involved in gene repression or by inhibiting the binding of transcription factors to DNA . HoweverTo further explore the mechanism of MNDA down-regulation in LA, we predicted that SPI1 is the transcriptional regulator of MNDA, that SPI1 is significantly down-regulated in lung cancer, and that MNDA expression increases with the increase in SPI1 expression. In addition, chipseq data also showed that SPI1 strongly binds to the promoter of MNDA in multiple cell lines. This suggests that the down-regulation of MNDA in LA may be caused by the down-regulation of its upstream transcription factor SPI1, which leads to a decrease in its transcription.miRNA is involved in many physiological processes such as cell proliferation, differentiation, apoptosis, and tissue and organ formation , and plaThis study has several limitations. First, this experiment mined the expression and prognostic significance of MNDA through online public databases; therefore, further studies with clinical samples are needed to validate these results. Second, we preliminarily explored the immune-related functions of MNDA in LA, and in vitro and in vivo experiments need to be designed to further study the detailed mechanism of MNDA involved in tumor immunity in lung cancer. Third, although we predicted the mechanism of MNDA expression regulation using bioinformatics methods, more powerful evidence to prove the accuracy of our conclusions is still needed.MNDA can be used as a novel and valuable biomarker for the diagnosis and treatment of LA. The abnormal expression of MNDA in LA is the result of the combined effects of transcriptional and post-transcriptional regulation.


Moraceae family with antioxidant and anti-inflammatory properties; however, the blood–brain barrier (BBB) prevents this flavonoid from reaching the CNS when aiming to potentially treat AD. Seeking to use the LAT-1 transporter present in the BBB, a nanoparticle (NPs) formulation loaded with MH and functionalized with phenylalanine-phenylalanine dipeptide was developed (NPphe-MH) and compared to non-functionalized NPs (NP-MH). In addition, two formulations were prepared using rhodamine B (Rh-B) as a fluorescent dye (NPphe-Rh and NP-Rh) to study their biodistribution and ability to cross the BBB. Functionalization of PLGA NPs resulted in high encapsulation efficiencies for both MH and Rh-B. Studies conducted in Wistar rats showed that the presence of phenylalanine dipeptide in the NPs modified their biodistribution profiles, making them more attractive for both liver and lungs, whereas non-functionalized NPs were predominantly distributed to the spleen. Formulation NPphe-Rh remained in the brain for at least 2 h after administration.Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder, with its incidence constantly increasing. To date, there is no cure for the disease, with a need for new and effective treatments. Morin hydrate (MH) is a naturally occurring flavonoid of the  Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder, with its incidence constantly increasing as elderly populations grow worldwide. Currently, more than 55 million people live with this disease worldwide, with nearly 10 million new cases every year ,3.Moraceae family with antioxidant and anti-inflammatory properties that could be a potential candidate for the treatment of neurodegenerative diseases such as Parkinson’s disease (PD) [Morin hydrate (MH) is a naturally occurring flavonoid of the ase (PD)  and Alzhase (PD) . Its potase (PD) ,7, also ase (PD) ,9. Both ase (PD) . In addiase (PD) . Howeverase (PD) ,13. In aase (PD) ,15.Nanosystems such as polymeric nanoparticles (NPs) prepared with PLGA are one of the approaches used to facilitate the passage of drugs across the BBB ,17,18, aThe BBB is a complex and efficient barrier that prevents foreign substances from entering the CNS. Small endogenous molecules such as amino acids, glucose, and others that are necessary for the survival of brain cells, use specific transporters expressed at the luminal side of the endothelial cells present in the BBB . One of Phenylalanine (phe) is a naturally occurring amino acid that can be found in proteins. As a substratum of LAT-1, phenylalanine may be an interesting option as a targeting ligand for the development of polymeric NPs destined to facilitate the passage of drugs across the BBB when treating CNS disorders ,30.In this work we aimed to develop and characterize a new formulation of biodegradable MH nanoparticles functionalized with phenylalanine (phe-phe) dipeptide to facilitate their passage across the BBB and remain in the CNS for as long as possible. For this, two types of MH-loaded NPs were developed using PLGA as polymer: non-functionalized MH-loaded PLGA NPs and PLGA:PLGA-phe-phe-functionalized MH-loaded NPs. To study their biodistribution and ability to cross the BBB in rats, rhodamine B (Rh-B), a fluorescent dye unable to cross the BBB, was used for the development of Rh-B-loaded PLGA NPs and PLGA:PLGA-phe-phe-functionalized Rh-B-loaded NPs.® RG 502 with a ratio of 50:50 poly (D.L-lactic-co-glycolic) acid  was obtained from Evonik Industries AG . Dichloromethane (DCM), polyvinyl alcohol  and dimethyl sulfoxide (DMSO) were provided by Sigma-Aldrich Química, S.A. . Distilled and deionized water  was used in the preparation of all buffers and solutions. For 1H NMR analysis all reagents were purchased from Sigma-Aldrich Química, S.A.  and Fluka . Solvents were obtained from SDS . All solvents and reagents were of analytical grade and were used as received without further purification.Morin hydrate (MH), rhodamine B (Rh-B), phenylalanine dipeptide (phe-phe), N-hidroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) were obtained from Sigma-Aldrich Química, S.A. . PLGA Resomerv/v) to remove residual by-products. The solid was diluted with DCM (15 mL), dried under anhydrous sodium sulfate, and filtered. The solvent was removed under vacuum in a room-temperature water bath. Finally, the solid was freeze-dried  to obtain PLGA-NHS (2.2 g) as a white powder. The spectrum obtained was 1H NMR  δ 5.33–5.16 , 4.97–4.71 , 2.86 , and 1.62 .PLGA-COOH  was dissolved in dry DCM (7 mL), converted to PLGA-NHS with an excess of N-hydroxysuccinimide  in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide , and then left to stirred for 22 h at room temperature under argon atmosphere. The resulting solution was added dropwise to ice-cooled ethyl ether (10 mL), and the solid was decanted and repeatedly washed in an ice-cooled mixture of ethyl ether and methanol . The solid was decanted and repeatedly washed with the same solvent to remove unreacted peptides and residual by-products. Finally, the solid was freeze-dried to obtain PLGA-phe-phe (120 mg) as a white powder. The spectrum obtained was 1H NMR  δ 7.17 , 5.20 , 4.82 , 1.58 , and 1.28 .PLGA-NHS  and dipeptide phe-phe  were dissolved in 12 mL of dry DCM and then N, N-diisopropylethylamine  was added. The mixture was gently stirred for 40 h under an argon atmosphere. The crude reaction was concentrated to dryness under vacuum without external heating. The resulting solution was washed with 15 mL of ice-cooled petroleum ether/methanol co-solvent  with fluorescent indicator (SDS CCM221254) and visualized with a UV lamp. For proton magnetic resonance spectroscopy (H1 NMR) spectra were acquired using a Bruker Avance 250 spectrometer working at 250 MHz for 1H and 63 MHz for 13C and operated via the standard Bruker software . Chemical shifts are reported as parts per million (ppm) relative to tetramethylsilane, and spin multiplicities are given as s (singlet), d (doublet), t (triplet), q (quartet), or m (multiplet).Morin-loaded PLGA nanoparticles (Rhodamine B-loaded PLGA NPs (formulation NP-Rh) were also prepared by the same method and used in biodistribution studies. Briefly, 2.5 mg of Rh-B and 50 mg of PLGA 502 were dissolved in DCM (2 mL). This solution was added dropwise into 6 mL of 1.5% PVA and sonicated. The organic solvent was removed. The NPs were then washed, centrifuged, and freeze-dried under the same conditions. Blank PLGA NPs were also prepared (formulation NP-0). All formulations were prepared in triplicate.Formulation NPphe-MH was prepared as NP-MH using 50 mg of PLGA:PLGA-phe-phe (60:40 ratio) instead of PLGA , which wFormulation NPphe-Rh was prepared as NP-Rh using PLGA:PLGA-phe-phe (60:40 ratio) instead of PLGA . For thiThe shape and surface morphology of the NPs were analyzed by scanning electron microscopy . Samples were coated with a thin layer of colloidal gold applied in a cathodic vacuum evaporator before observation in a microscope.® Ultra 3500 system . The diameter was expressed as volume diameter. The polydispersity index (PDI) was also calculated.The mean diameter and size distribution of the NPs were determined by laser diffraction using a Zetatrac® . Measurements were carried out at 25 °C in aqueous solution. For this, 5 mg of each formulation was suspended in 50 mL of distilled water and stirred for 1 min. Then, the aqueous dispersion of NPs at a concentration of 100 µg/mL was placed in a capillary cell  for zeta potential measurements. All formulations were analyzed in triplicate.Zeta potential was determined by Laser-Doppler anemometry using a Malvern Zetasizer Nano Sv/v). The flow rate was 1.4 mL/min, and the volume injected was 50 µL. The detection wavelength was 256 nm. All analyses were carried out at 40 ± 0.5 °C. Drug loading (DL) was also determined by HPLC and expressed as the amount of MH incorporated by 100 mg of NPs.Encapsulation efficiency (EE%) of MH within the NPs was determined as follows: 10 mg of each formulation was dissolved in 1 mL of DCM. Then, 15 mL of methanol was added to this solution and centrifuged for 5 min at 5000 rpm. The supernatant (5 mL) was mixed with 5 mL of HCl 0.1 N and analyzed by HPLC . The equTo determine the EE% and DL of Rh-B, the same procedure was used. Quantification was performed by spectrophotometry at 555 nm.In vitro release studies were carried out in a Memmert WB22 water bath  at 37 ± 1 °C and constant agitation (100 rpm). For this, 20 mg of NPs were suspended in PBS at pH 7.4 (5 mL). At regular time intervals, samples were centrifuged with the supernatant withdrawn and filtered through 0.45 µm filters. Quantification of MH was performed by HPLC . QuantifMale Wistar rats  weighing 220–270 g were housed with controlled temperature and a 12 h:12 h light:dark cycle, observed daily, and fed with a commercial pelleted diet and water ad libitum. All animal procedures were approved by the Ethics Committee on Animal Experimentation  and the regional authorities . All procedures complied with the European Community Council Directive (010/63/UE). Efforts were made to minimize the number of animals used and their suffering.-n = 4) received saline solution.Group 1 (-n = 8) received formulation NP-Rh.Group 2 (-n = 6) received formulation NPphe-Rh.Group 3 . All organs were kept at −80 °C prior to analysis. To observe the biodistribution of NPs prepared with Rh-B, a portion (1.5 ± 0.1 g) of each organ  was homogenized using a turbine with 5 mL of DCM. After 1 h, the mixture was centrifuged at 8000 rpm for 10 min to extract all Rh-B content. Rh-B was quantified by measuring its fluorescence in a Varian Cary Eclipse fluorescence spectrophotometer . The procedure used was that described by Marcianes et al.  and adapThe amount of Rh-B was expressed as ng of Rh-B/g of tissue. As the different formulations showed different drug loading, the results were normalized for the same dose of Rh-B.To study the passage of NPs through the BBB, brains were divided into both hemispheres. The right hemispheres were sectioned into 25 µm slices, embedded for 24 h in DPX  + polystyrene (25 g) + xylene (70 mL)), stained with DAPI solution  (125 ng/mL) , and anaThe left hemispheres were homogenized with DCM (5 mL) and centrifuged at 8000 rpm for 10 min to extract all Rh-B content. The amount of Rh-B was expressed as ng of Rh-B/g of tissue.® Centurion software . A p-value < 0.05 was considered statistically significant.Results were expressed as mean ± standard deviation. Multifactorial ANOVA tests were performed using the Statgraphics 19-X64The surface modification of nanosystems can be carried out for several purposes: to improve the stability of polymeric NPs, increase their residence time within the body, reduce aggregation, improve their passage through biological barriers, and/or achieve cellular internalization . At the level of the BBB, multiple transporters are expressed, influencing the barrier permeability for various small molecules, such as amino acids, glucose, and iron transferrin . In thisOn the other hand, amyloid fibrils are the hallmark of several diseases, including Alzheimer’s disease ,41. RechFor these reasons, we have developed a novel nanosystem in the form of polymeric NPs functionalized with phenylalanine dipeptide (phe-phe). When attaching surface ligands to nanosystems, some authors have indicated that functionalization of the polymer prior to the elaboration of the NPs is quite effective as it allows for adequate control of the surface decoration .In our study, the -CO2H groups of PLGA were activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The activated polymer was purified, with activation being confirmed by proton magnetic resonance spectroscopy (H1 NMR). Then, activated PLGA reacted with the phe-phe peptide, and functionalization was confirmed by H1 NMR with the presence of aromatic signals at 7.2 ppm and missing characteristic NHS group signals at 2.9 ppm. The conjugation efficiency obtained was around 80%  .p < 0.05) were only found when compared to unloaded NPs. Particle sizes under 200 nm will allow the nanosystems to cross the BBB [As shown in  the BBB ,45,46. TAll formulations presented PDI ≤ 0.3 , indicatAll formulations exhibited negative zeta potential values. Particles with zeta potentials of more than +30 mV or more than −30 mV are normally considered stable with a low tendency for aggregation. Positively charged NPs may cause adverse systemic effects, such as hemolysis. On the contrary, negative or neutral charges appear to show longer circulating times and fewer side effects .When comparing zeta potential values of blank NPs (NP-0 vs. NPphe-0), the effect of the presence of phenylalanine was clear. Formulation NP-0 exhibited a value lower than −20 mV (−25.4 ± 1.4 mV), as expected due to the carboxylic acid groups present in PLGA. Other authors have also obtained similar results ,50. RegaDifferences in zeta potential were also found between non-functionalized and phe-phe functionalized MH-loaded NPs. In NP-MH, the flavonoid increased the zeta potential to −20.8 ± 0.5 mV, which was slightly lower in NPphe-MH (−23.3 ± 0.7 mV). This could be explained by the fact that in NPphe-MH, not all the carboxylic acid groups were expressed on the surface of the particles. If we compare the values of EE%, functionalization of NP-MH with the dipeptide (formulation NPphe-MH) led to higher encapsulating efficiency, as some of the phe moieties could be interacting with the flavonoid, favoring its affinity for the polymeric matrix, thereby leading to higher EE%. If so, these phe moieties would not be present on the surface of the NPs.From these findings, it would be expected to achieve similar results with the Rh-B formulations. However, Rh-B-loaded NPs (NP-Rh and NPphe-Rh) showed the greatest differences in zeta potential (−22.6 ± 0.4 mV vs. −7.88 ± 0.2 mV). For NPphe-Rh, zeta potential markedly increased. Rh-B is positively charged, and the EE% of NPphe-Rh was two-fold that of NP-Rh. The presence of the fluorescent dye on the surface of the NPs could account for this variability. This is supported by data obtained from the in vitro release tests in which formulation NPphe-Rh exhibited the highest release of the fluorescent dye within the first hour.High EE% values (>80%) were obtained for all MH-loaded NPs and especially for formulation NPphe-MH with a mean value of 96.5 ± 3.2% . SimilarRegarding EE% of Rh-B within PLGA NPs, low values were obtained (<20%); however, formulation NPphe-Rh resulted in two-fold EE% than NP-Rh. This may be explained by the fact that Rh-B is freely dissolved in water. The higher EE% found in both MH- and Rh B-loaded functionalized PLGA NPs may be attributed to the interaction of aromatic phe moieties present in the polymer with aromatic rings of MH and Rh-B, which favors their affinity for the polymeric matrix, thereby increasing EE% of both agents. In vitro release tests led to similar profiles during the first 24 h for non-functionalized and functionalized MH-loaded NPs . Mean buThereafter, the zero-order release kinetics of MH was observed for both formulations up to 192 h. During this time, NPs prepared with the amino acid (NPphe-MH) showed a slightly faster release of the flavonoid. With this formulation, therapeutic concentrations could be theoretically maintained for 200 h. In fact, the therapeutic concentrations of MH when potentially treating AD do not necessarily need to be high. Alberdi et al.  found thIn vitro release tests were also conducted for Rh-B-loaded NPs to clarify the release behavior of Rh-B during the first two hours, taking into consideration that these formulations have been only developed for the in vivo biodistribution and BBB studies. As these studies have been carried out 1 h and 2 h after administration, our aim was to evaluate the in vitro release of Rh-B from the NPs at these times. Mean Rh-B release percentages of 44.00 ± 0.35% and 55.04 ± 0.44% were obtained for formulations NP-Rh and NPphe-Rh, respectively, at time 2 h. p-value = 0.005). In addition, statistically significant differences resulted between both formulations  in all the tissues analyzed. As non-statistically significant differences were found when both times (1 h and 2 h) were analyzed together with the other variables, additional independent ANOVA tests were carried out to analyze the effects of time, organ distribution, and formulation (Biodistribution studies were performed for both Rh-B-loaded PLGA NPs (NP-Rh and NPphe-Rh), with fluorescence being detected in the liver, kidneys, spleen, and lungs of Wistar rats. Multifactorial ANOVA tests were applied to the data considering experimental variables formulation, organ biodistribution, and time. From the results obtained, statistically significant differences in organ distribution were found . Regarding NPphe-Rh, the greatest differences in biodistribution were found in the liver and lungs when comparing both times. Indeed, a statistically significant  increase in the liver and decrease in the lungs was found at 2 h when compared to 1 h. As occurred at time 1 h, the non-functionalized NPs preferentially accumulated in the spleen, followed by the liver and lungs, whereas functionalized NPs showed higher affinity for the liver and lungs and low affinity for the spleen.At time 2 h, the accumulation of formulation NP-Rh in the spleen was significantly reduced with respect to 1 h (It is known that PLGA NPs mainly accumulate in the liver and spleen after i.v administration due to sequestration by cells of the mononuclear phagocytic system (MPS), with particle size and surface characteristics being key parameters influencing their biodistribution . MohammaIn our study, the liver was the organ of highest deposition, followed by the spleen and lungs . This reIn our study, the effect of endogenous transporters on the biodistribution of NPs was not analyzed. As particles sizes of NP-Rh and NPphe-Rh were similar, differences found in biodistribution could be, at least partially, attributed to differences in their surfaces, as the presence of phenylalanine in the polymer matrix might modify the biodistribution of NPs at the studied time points.To investigate the ability of NPs to cross the BBB, brains were removed after sacrifice and divided into both hemispheres. Images obtained from one of the hemispheres clearly showed the presence of Rh-B , even thp-value > 0.05). However, at 2 h, the intensity of fluorescence from formulation NPphe-Rh was significantly higher. This might indicate that the presence of the dipeptide (phe-phe) in the formulation increased the passage of NPs through the BBB at longer times or, at least, prevented them from being expelled by means of the P-glycoprotein (P-gp) efflux pump, a transport mechanism, which is usually involved [In addition, the quantification of the fluorescence intensity of Rh-B in brain samples was performed using ImageJ software A. At timinvolved .p-value > 0.05). However, at time 2 h, the amount of Rh-B was significantly higher for formulation NPphe-Rh in comparison to NP-Rh. The similar results obtained by both analytical techniques (microscopy and fluorometry) seem to indicate that attaching phenylalanine to the NPs surfaces increased their residence time within the CNS. It is known that this amino acid shows a favorable gradient to access the brain without being expelled , which cThe results obtained at 2 h are very encouraging, taking into consideration that when developing nanosystems targeting the CNS, the activity of the P-gp efflux pump is a drawback that needs to be overcome. Rh-B is a known P-gp substrate; therefore, once released from the NPs, it will be expelled from the brain. For this, the results obtained indicate that either the NPphe-Rh NPs crossed more slowly the BBB but to a greater extent than NP-Rh or remained in the brain for a longer period while releasing Rh-B, whereas non-functionalized NPs would have been already expelled by the efflux pumps.Limitations of our research include the small number of animals tested, high interindividual variability, and the difficulty of predicting the in vivo release of MH from the results obtained in the in vitro release tests conducted with the NPs. The nanoparticulate formulations developed were adequate for encapsulating MH flavonoid; however, when using the phenylalanine dipeptide in the elaboration of NPs, an increase in the encapsulation efficiency of MH and Rh-B was obtained. As both molecules possess aromatic rings in their structures, the increases obtained may be attributed to the interaction between these aromatic rings and that of phenylalanine, leading to a higher affinity of MH and Rh-B for the polymeric matrix of the NPs. The presence of phenylalanine dipeptide might have contributed to modifying the biodistribution profile of the NPs, making them more attractive for the liver and lungs. On the contrary, non-functionalized NPs were predominantly distributed to the spleen. Nevertheless, more research should be conducted to investigate the effects of endogenous transporters on the biodistribution profiles of the NPs. Linkage of phenylalanine dipeptide to PLGA in formulation NPphe-Rh resulted in an increased presence of the NPs in brain tissue at longer times (2 h) which agrees with the affinity of the CNS for the amino acid.


Rheumatoid arthritis (RA) is a disease characterized by a chronic inflammatory state. High pro-inflammatory cytokine levels are associated with disease activity. Exercise and the Mediterranean diet (MD) exert anti-inflammatory effects; however, their impacts on inflammation in RA patients remains unknown. This study aimed to compare the effects of six-months of dynamic exercise program (DEP) vs. MD on pro- and anti-inflammatory cytokine serum concentrations.n = 30), MD (n = 30), or control group (n = 30). All patients received pharmacological treatment. Serum concentrations of pro-inflammatory  and anti-inflammatory  cytokines were measured at baseline and after 6 months using the Luminex technique.Secondary analysis of a randomized clinical trial in which 90 women with RA were randomly assigned to the DEP (p = 0.01), TNF-β , and IL-6  in the DEP and MED groups in comparison with control group. IL-1Ra concentrations increased only in the MD group (13.8) compared to levels in the control group (−31.7), p = 0.04. There were no statistically significant differences between DEP and MD groups. Only n = 27 participants in the DEP group, n = 26 in the MD group, and n = 21 in the control group completed the follow-up.After 6 months of follow-up, we found an improvement of the median percentages changes concentrations of TNF-α (DEP, −12.3; MD, −13.3; control, 73.2; The DEP and the MD have potential effects in the concentrations of pro-inflammatory cytokines compared with those in a control group. Only the MD elevated the concentration of IL-Ra.ClinicalTrials.gov], identifier [NCT02900898].[ Rheumatoid arthritis (RA) is a chronic disease of an autoimmune nature characterized by the destruction of the synovial membrane, inflammation, and joint pain . This diIn the pathogenesis of RA, chronic inflammation of the synovial membrane occurs by the infiltration of T and B lymphocytes, endothelial cells, and macrophages that produce cytokines . They are usually classified as pro-inflammatory or anti-inflammatory. In RA, high cytokine concentrations have been associated with disease activity, structural joint damage, and poor prognosis. TNF-α, IL-1β, and IL-6 are among the most relevant pro-inflammatory cytokines in the pathogenesis of this disease –6. It haPharmacological and non-pharmacological treatments aim to control symptoms and dampen the immune response . Non-phaPrevious studies have reported the benefits of exercise in RA patients , 13. WheOn the other hand, the Mediterranean diet (MD) is characterized by the incorporation of large amounts of vegetables, fruits, cereals, and legumes. Olive oil is a significant source of fat in this diet, whereas dairy products are included mainly in the form of yogurt and cheese in moderate amounts. This diet is distinguished for being low in saturated fat but high in fiber, complex carbohydrates, monounsaturated fatty acids, and antioxidant vitamins . SeveralWe conducted a secondary analysis of a randomized clinical trial that included consecutive women with RA diagnosis. We evaluated the individual effect of a DEP or the MD on serum concentrations of pro-inflammatory cytokines  and anti-inflammatory cytokines (IL-10 and IL-1Ra) after 6 months of intervention. We hypothesized that in women with RA, 6 months of DEP or MD therapy would decrease the concentrations of TNF-α, TNF-β, IL-1β, and IL-6 compared to those in the control group.ClinicalTrials.gov (ID: NCT02900898) that was undertaken from August 2016 to December 2019. Between June and July 2020, we performed this secondary analysis. The rationale for the trial design details and eligibility features, as well as the main results, have been published previously  diagnosed with RA according to the 2010 criteria of the American College of Rheumatology/European League Against Rheumatism 8 years d were recAll patients signed written informed consent prior to enrollment, and the Institutional Human Ethics and Research Committees approved the study (Ref: 1347).Sample size was calculated using a multiple comparisons formula, which considered 95% accuracy and 80% power, according to the IL-6 mean change from previous studies , resultiwww.randomization.com to one of three groups: 1) DEP (n = 30), 2) MD (n = 30), and control group (n = 30). An external collaborator created the randomization sequence, and the researchers carried out the assignment process.Participants were randomly assigned using a sequence created by the website ® version 11.1. Patients randomized to the MD group received general physical activity recommendations.Full details of the MD dietary intervention have been previously published . It consThe DEP intervention has been described previously  see Sup. It consParticipants assigned to the control group received verbal recommendations concerning physical activity guides and basic nutrition information , 26, 27.The primary outcome of this secondary analysis consisted of the differences in the cytokine concentration changes among the three groups.An independent investigator who was blinded to the group assignment of the patients performed the cytokine analysis. At baseline, 5-mL fasting blood samples were collected 1 week before the beginning of the interventions. Post-intervention measurement of cytokines was performed 72 h after the last exercise session in the DEP group and 6 months after the basal measurement in the MD and control groups. All samples were stored in gel serum separator tubes and centrifuged at 3,000 rpm for 15 min.® technique according to the manufacturer’s instructions. Reading of plates was performed using Bio-Plex 200 equipment  with the Bio-Plex Manager Software. Cytokine concentrations were determined by evaluating the average fluorescence intensities in a calibration curve.Cytokine quantification was performed using the Human Cytokine kit  by means of the LuminexTwo rheumatologists (blinded to group assignments) collected all clinical data related to RA, including symptoms, disease duration, associated comorbidities, and pharmacological treatment, and calculated disease activity score-28 (DAS28) values . Acute pPost hoc comparisons were performed using the Mann–Whitney U test with Bonferroni correction. All analyses were performed using SPSS Version 25 , two-tailed tests were used, and a p-value <0.05 was considered statistically significant.The Shapiro–Wilk test was used to assess the normality of the variables. Data are reported as the mean ± standard deviation (SD) if normally distributed or as median and percentiles 25–75 (p25-p75) if otherwise, whereas categorical variables are presented as frequencies and percentages. The Wilcoxon test was used to evaluate the changes from baseline to final measurements within each group. For the post-intervention comparison of groups, percentage changes were calculated and analyzed with the Kruskal–Wallis test. A total of 90 women with RA were included in this secondary analysis and were randomly assigned to the three intervention groups (30 patients per group), as shown in 2. All patients showed low disease activity (DAS28 <3.2). Baseline demographic and clinical characteristics for each study group are presented in The mean age of all patients was 46.2 ± 11.5 years, with a BMI of 25.8 kg/mn = 27), 4 (13.3%) in the MD group (n = 26), and 9 (30%) in the control group (n = 21) did not complete the follow-up. Reasons included work issues or unknown causes (see Three of the participants (10%) in the DEP group (uses see . Baselinp = 0.06). In the DEP group, exercise adherence was 72%.Before study enrollment, 14 (15.2%) patients reported that they performed physical activity, being this activity more frequent in patients in the DEP group (23.3%), followed by the MD group (20%), and <3% in the control group, without a significant difference between groups (p = 0.01), while in the DEP (4 to 4.4) and control groups (4.35 to 3.82) this score did not show a significant change.In the MD group, the MedScore changed from 4.6 to 5.7 (p = 0.13).Disease activity score-28 did not change, delta in DEP [median ] was −0.05 , MD 0.22 , control −0.23  and there were not statistical significant differences between groups (p = 0.04). IL-6 also showed this behavior after the intervention, with a decrease from 4.3 to 2.1 pg/mL (p = 0.01) in the MD group. No other significant intragroup changes were found after 24 weeks of intervention.As shown in p = 0.01). Similarly, TNF-β decreased in the DEP group (−67.4%) and the MD group (−54.9%), while the control group exhibited no changes at the end of the study, with a significant difference among the control and intervention groups (p = 0.04). There were, however, no statistically significant differences between DEP and MD groups. On the other hand, the MD group showed a statistically significant difference in IL-1Ra (13.8%) concentration compared to the control group (−31.7%), p = 0.04. The other cytokines that were evaluated did not show differences among groups after 24 weeks of intervention. Regarding differences in baseline cytokine concentrations between patients who completed and those who did not finish the follow-up, we observed differences in the cytokines TNF-β and IL-1Ra  and compared them with healthy participants (n = 47). No changes were found in any study group in IL-1β, TNF-α, or IL-6 levels  (p < 0.001) and IL-6  in the MD group (n = 3042) that those with greater adherence to the MD exhibited concentrations of IL-6 17% (p = 0.02) lower than in those with poorer adherence. In contrast, no differences were found in TNF-α concentrations  consumed in this diet . The cur< 0.001) . MoreoveMD group . Additiotrations . Finallyup n = 30 that thotrations .It is important to note that the term immunometabolism has been used recently, since the immune system monitors and responds to specific metabolic changes and is very sensitive to environmental changes to maintain systemic homeostasis. Diet and physical fitness status are the main environmental factors that affect the function of the immune system. An excess or deficiency in nutrients alters the immune response; therefore, the inclusion of the metabolic profile and the physical status, as well as the interaction between these factors, is essential in studies where inflammatory markers are evaluated , 45.We found differences in the baseline cytokine concentrations of the participants who completed the follow-up and those who abandoned the study see ; this isThe present study has some limitations. First, our results are not conclusive because it is a secondary analysis, so further research is needed with larger sample sizes and robust methodologies. Second, the loss of participants in the control group exceeded 10%, which could have affected the results. Third, the metabolic profile  was not assessed, and therefore, it was difficult to understand the inflammatory profile of the participants. Fourth, there is limited generalizability of the results given that only women were included and, more importantly, a group of healthy women was not entered as a control to have a comparison value of the anti- and pro-inflammatory cytokines studied. Unfortunately, at present, there are no standardized reference values for these molecules. Fifth, some of the results are based on the estimation of diet based on a questionnaire. Finally, the pharmacologic treatment effect on the cytokine profile was not assessed, as a control group without pharmacologic treatment was not evaluated.The outcome of the present study suggests that women with RA with low disease activity after 24-week intervention of a DEP or an MD may decrease the percentage change of serum concentrations of pro-inflammatory cytokines  compared to the control group. In contrast, the consumption of an MD increases the percentage change of serum concentrations of the anti-inflammatory cytokine IL-1Ra compared to the control group.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Instituto Nacional de Ciencias Médicas y Nutrición SZ Human Ethics and Research Committees. The patients/participants provided their written informed consent to participate in this study.JP-J, LC-M, JA-V, and LL designed the study. ML-M, LC-M, and MO-M analyzed data and wrote the manuscript. AH-A performed clinical evaluation of the patients. MO-M and JG-M implemented nutritional and exercise protocols. ML-M and MO-M collected the data. AH-A and LL edited the manuscript. All authors have reviewed the manuscript and approved its submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.